Human vascular IBP-like growth factor

ABSTRACT

A human Vascular IBP-Like Growth Factor polypeptide (VIGF) and DNA (RNA) encoding such polypeptide and a procedure for producing such polypeptide by recombinant techniques is disclosed. Also disclosed are methods for utilizing such polypeptide for wound healing or tissue regeneration, stimulating implant fixation and angiogenesis. Antagonists against such polypeptides and their use as a therapeutic to treat atherosclerosis, tumors and scarring are also disclosed. Diagnostic assays for identifying mutations in VIGF nucleic acid sequences and altered levels of the VIGF polypeptide are also disclosed.

This application is a continuation-in-part of PCT/U.S. Ser. No.94/14388, filed Dec. 9, 1994.

This application is a continuation-in-part of PCT/U.S. Ser. No.94/14388, filed Dec. 9, 1994.

This invention relates to newly identified polynucleotides, polypeptidesencoded by such polynucleotides, the use of such polynucleotides andpolypeptides, as well as the production of such polynucleotides andpolypeptides. The invention also relates to inhibiting the action ofsuch polypeptides.

The polypeptide of the present invention is related to a family ofgrowth regulators comprising cef 10/cyr 61, connective tissue growthfactor (CTGF), and nov, as well as the insulin-like growth factorbinding protein (IBP) family which modulates the activity ofinsulin-like growth factor (IGF). The mRNA corresponding to thepolypeptide of this invention is highly expressed in vascularcell-types, thus, this polypeptide is hereinafter referred to as humanvascular IBP-like growth factor or "VIGF".

Growth factors and other mitogens, including transforming oncogenes, arecapable of rapidly inducing a complex set of genes to be expressed bycertain cells (Lau, L. F. and Nathans, D., Molecular Aspects of CellularRegulation, 6:165-202 (1991). These genes, which have been namedimmediate early or early response genes, are transcriptionally activatedwithin minutes after contact with a growth factor or mitogen,independent of de novo protein synthesis. A group of these immediateearly genes encodes secreted, extracellular proteins which are neededfor coordination of complex biological processes such as differentiationand proliferation, regeneration and wound healing (Ryseck, R. P. et al,Cell Growth Differ., 2:235-233 (1991).

Highly related proteins which belong to this group include cef 10 fromchicken, which was detected after induction by the viral oncogenepp60^(v-src) (Simmons, D. L. et al, PNAS, U.S.A., 86:1178-1182 (1989). Aclosely related protein, cyr 61, is rapidly activated by serum orplatelet-derived growth factor (PDGF) (O'Brien, T. P. et al, Mol. CellBiol., 10:3569-3577 (1990). The overall amino acid identity between cef10 and cyr 61 is as high as 83%. A third member is human connectivetissue growth factor (CTGF) (Bradham, D. M. et al., J. Cell. Biol.,114:1285-1294 (1991). CTGF is a cysteine-rich peptide which is secretedby human vascular endothelial cells in high levels after activation withtransforming growth factor beta (TGF-β). CTGF exhibits PDGF-likebiological and immunological activities and competes with PDGF for aparticular cell surface receptor.

A fourth member of the immediate-early proteins is fisp-12, which hasbeen shown to be induced by serum and has been mapped to a region of themurine genome (Ryseck, R. P. et al., Cell Growth Differ., 2:235-233(1991). Yet another member of this family is the chicken gene, nov,normally arrested in adult kidney cells, which was found to beoverexpressed in myeloblastosis-associated virus type 1 inducednephroblastomas. Further, expression of an amino-terminal-truncated novproduct in chicken embryo fibroblasts was sufficient to inducetransformation (Joliot, V. et al., Mol. Cell. Biol., 12:10-21 (1992).

The expression of these immediate early genes act as "third messengers"in the cascade of events triggered by growth factors. It is also thoughtthat they are needed to integrate and coordinate complex biologicalprocesses, such as differentiation and wound healing in which cellproliferation is a common event.

This emerging family of growth regulators is called the CCN family forCTGF; cef 10/cyr 61; and nov. The VIGF polypeptide of the presentinvention is thought to be a member of this family of growth regulators.The VIGF polypeptide also contains a stretch of cysteines which ishighly homologous to insulin-like growth factor (IGF)-binding protein.

At least two different binding proteins have been identified in adulthuman serum, namely, IGF-binding protein 53 and IGF-binding protein 1.The IGF-binding proteins have both stimulatory and inhibitory effects onIGF. Clemmons, et al, J. Clin. Invest., 77:1548 (1986) showed increasedbinding to fibroblast and smooth muscle cell surface receptors of IGF incomplex with its binding protein. The inhibitory effects of IGF-bindingprotein on various IGF actions in vitro, have been shown and theyinclude stimulation of glucose transport by adipocytes, sulfateincorporation by chondrocytes and thymidine incorporation in fibroblast(Zapf, et al., J. Clin. Invest., 63:1077 (1979)). In addition,inhibitory effects of IGF-binding proteins on growth factor mediatedmitogen activity in normal cells has been shown.

In accordance with one aspect of the present invention, there isprovided a novel mature polypeptide which is VIGF, as well asbiologically active and diagnostically or therapeutically usefulfragments, analogs and derivatives thereof.

In accordance with another aspect of the present invention, there areprovided isolated nucleic acid molecules encoding human VIGF, includingmRNAs, DNAs, cDNAs, genomic DNAs as well as analogs and biologicallyactive and diagnostically or therapeutically useful fragments andderivatives thereof.

In accordance with yet a further aspect of the present invention, thereis provided a process for producing such polypeptide by recombinanttechniques comprising culturing recombinant prokaryotic and/oreukaryotic host cells, containing a human VIGF nucleic acid sequence,under conditions promoting expression of said protein and subsequentrecovery of said protein.

In accordance with yet a further aspect of the present invention, thereis provided a process of utilizing such polypeptide, or polynucleotideencoding such polypeptide for therapeutic purposes, for example, totreat muscle wasting diseases, osteoporosis, to aid in implant fixation,to stimulate wound healing or tissue regeneration, to promoteangiogenesis and to proliferate vascular smooth muscle and endothelialcell production.

In accordance with yet a further aspect of the present invention, thereare provided antibodies against such polypeptides.

In accordance with yet another aspect of the present invention, thereare provided antagonists to such polypeptides, which may be used toinhibit the action of such polypeptides, for example, to limit theproduction of excess connective tissue during wound healing or pulmonaryfibrosis.

In accordance with yet a further aspect of the present invention, thereare also provided nucleic acid probes comprising nucleic acid moleculesof sufficient length to specifically hybridize to VIGF sequences.

In accordance with still another aspect of the present invention, thereare provided diagnostic assays for detecting diseases related to theunder-expression and over-expression of the VIGF polypeptide andmutations in the nucleic acid sequences encoding such polypeptide.

These and other aspects of the present invention should be apparent tothose skilled in the art from the teachings herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings are illustrative of embodiments of the inventionand are not meant to limit the scope of the invention as encompassed bythe claims.

FIG. 1(A-E) shows the cDNA (SEQ ID NO:1) and corresponding deduced aminoacid sequence (SEQ ID NO:2) of the VIGF polypeptide. The initial 21amino acids represent the putative leader sequence such that the maturepolypeptide comprises 163 amino acids. The standard one letterabbreviations for amino acids are used. Sequencing was performed using a373 Automated DNA sequencer (Applied Biosystems, Inc.). Seqeuncingaccuracy is predicted to be greater than 97% accurate.

FIG. 2 shows the amino acid sequence homology between VIGF and otherproteins which are members of the CCN family. FIG. 2, contains seven (7)comparative polypeptide sequences: ce10₋₋ chick (SEQ ID NO:11), cyr6₋₋mouse (SEQ ID NO:12), ctgf₋₋ human (SEQ ID NO:13), fisp₋₋ 12 (SEQ IDNO:14), nov₋₋ chick (SEQ ID NO:15), ibp₋₋ 3human (SEQ ID NO:16) andccn-4 (SEQ ID NO:17). The comparative polypeptide sequences aredesignated by one-letter amino acid codes.

In accordance with an aspect of the present invention, there is providedan isolated nucleic acid (polynucleotide) which encodes for the maturepolypeptide having the deduced amino acid sequence of FIG. 1 (SEQ IDNO:2) or for the mature polypeptide encoded by the cDNA of the clonedeposited as ATCC Deposit No. 75874 on Aug. 25, 1994.

The ATCC number referred to above is directed to a biological depositwith the ATCC, 12301 Parklawn Drive, Rockville, Md. 20852. The strain isbeing maintained under the terms of the Budapest Treaty and will be madeavailable to a patent office signatory to the Budapest Treaty.

A polynucleotide encoding a polypeptide of the present invention may beobtained from human umbilical vein and aortic endothelial cells, aorticsmooth muscle cells, and pulmonary artery. The polynucleotide of thisinvention was discovered in a cDNA library derived from human umbilicalvein endothelial cells. It is structurally related to the IBP and CCNfamilies. It contains an open reading frame encoding a protein of 184amino acid residues of which approximately the first 21 amino acidsresidues are the putative leader sequence such that the mature proteincomprises 163 amino acids.

The designation of VIGF as a hybrid member of both the CCN growth factorand IBP families was based primarily through conservation of amino acidsequences. Similarity of VIGF to the CCN family is inferred because ofthe 40-45% similarity over the entire polypeptide, 12 of a total of 18VIGF cysteines are conserved, and 94% identity with the IBP signature(GCGCCXXCAXXXXXXC) which is perfectly conserved in every member of theCCN family.

The VIGF polypeptide also has significant similarity to the IBP family.In two adjacent regions, amino acids 30-44 (IBP signature) and 55-69,there is at least 80% identity to the IBP family. These regions arecontained within the putative IGF binding domain of the IBPs. The humantissue and cell-type specific expression has been determined by Northernblot analysis. The 2.3-2.4 kb VIGF mRNA is localized in the adult lungand kidney as shown using the procedure of Example 4. VIGF geneexpression was undetectable in heart, brain, placenta, liver, skeletalmuscle, and pancreas. Cultured human umbilical vein endothelial andaortic smooth muscle cells are cell-types which express VIGF mRNA at ahigh level while dermal foreskin fibroblasts show a very low level.Together, these results indicate that VIGF is primarily of vascularorigin.

The polynucleotide of the present invention may be in the form of RNA orin the form of DNA, which DNA includes cDNA, genomic DNA, and syntheticDNA. The DNA may be double-stranded or single-stranded, and if singlestranded may be the coding strand or non-coding (anti-sense) strand. Thecoding sequence which encodes the mature polypeptide may be identical tothe coding sequence shown in FIG. 1(A-E) (SEQ ID NO:1) or that of thedeposited clone or may be a different coding sequence which codingsequence, as a result of the redundancy or degeneracy of the geneticcode, encodes the same mature polypeptide as the DNA of FIG. 1(A-E) (SEQID NO:1) or the deposited cDNA.

The polynucleotide which encodes for the mature polypeptide of FIG.1(A-E) (SEQ ID NO:2) or for the mature polypeptide encoded by thedeposited cDNA may include: only the coding sequence for the maturepolypeptide; the coding sequence for the mature polypeptide andadditional coding sequence such as a leader or secretory sequence or aproprotein sequence; the coding sequence for the mature polypeptide (andoptionally additional coding sequence) and non-coding sequence, such asintrons or non-coding sequence 5' and/or 3' of the coding sequence forthe mature polypeptide.

Thus, the term "polynucleotide encoding a polypeptide" encompasses apolynucleotide which includes only coding sequence for the polypeptideas well as a polynucleotide which includes additional coding and/ornon-coding sequence.

The present invention further relates to variants of the hereinabovedescribed polynucleotides which encode for fragments, analogs andderivatives of the polypeptide having the deduced amino acid sequence ofFIG. 1(A-E) (SEQ ID NO:2) or the polypeptide encoded by the cDNA of thedeposited clone. The variant of the polynucleotide may be a naturallyoccurring allelic variant of the polynucleotide or a non-naturallyoccurring variant of the polynucleotide.

Thus, the present invention includes polynucleotides encoding the samemature polypeptide as shown in FIG. 1(A-E) (SEQ ID NO:2) or the samemature polypeptide encoded by the cDNA of the deposited clone as well asvariants of such polynucleotides which variants encode for a fragment,derivative or analog of the polypeptide of FIG. 1(A-E) (SEQ ID NO:2) orthe polypeptide encoded by the cDNA of the deposited clone. Suchnucleotide variants include deletion variants, substitution variants andaddition or insertion variants.

As hereinabove indicated, the polynucleotide may have a coding sequencewhich is a naturally occurring allelic variant of the coding sequenceshown in FIG. 1(A-E) (SEQ ID NO:1) or of the coding sequence of thedeposited clone. As known in the art, an allelic variant is an alternateform of a polynucleotide sequence which may have a substitution,deletion or addition of one or more nucleotides, which does notsubstantially alter the function of the encoded polypeptide.

The present invention also includes polynucleotides, wherein the codingsequence for the mature polypeptide may be fused in the same readingframe to a polynucleotide sequence which aids in expression andsecretion of a polypeptide from a host cell, for example, a leadersequence which functions as a secretory sequence for controllingtransport of a polypeptide from the cell. The polypeptide having aleader sequence is a preprotein and may have the leader sequence cleavedby the host cell to form the mature form of the polypeptide. Thepolynucleotides may also encode for a proprotein which is the matureprotein plus additional 5' amino acid residues. A mature protein havinga prosequence is a proprotein and is an inactive form of the protein.Once the prosequence is cleaved an active mature protein remains.

Thus, for example, the polynucleotide of the present invention mayencode for a mature protein, or for a protein having a prosequence orfor a protein having both a prosequence and a presequence (leadersequence).

The polynucleotides of the present invention may also have the codingsequence fused in frame to a marker sequence which allows forpurification of the polypeptide of the present invention. The markersequence may be a hexa-histidine tag supplied by a pQE-9 vector toprovide for purification of the mature polypeptide fused to the markerin the case of a bacterial host, or, for example, the marker sequencemay be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells,is used. The HA tag corresponds to an epitope derived from the influenzahemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).

The term "gene" means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as intervening sequences(introns) between individual coding segments (exons).

Fragments of the full length gene of the present invention may be usedas a hybridization probe for a cDNA library to isolate the full lengthcDNA and to isolate other cDNAs which have a high sequence similarity tothe gene or similar biological activity. Probes of this type preferablyhave at least 30 bases and may contain, for example, 50 or more bases.The probe may also be used to identify a cDNA clone corresponding to afull length transcript and a genomic clone or clones that contain thecomplete gene including regulatory and promotor regions, exons, andintrons. An example of a screen comprises isolating the coding region ofthe gene by using the known DNA sequence to synthesize anoligonucleotide probe. Labeled oligonucleotides having a sequencecomplementary to that of the gene of the present invention are used toscreen a library of human cDNA, genomic DNA or mRNA to determine whichmembers of the library the probe hybridizes to.

The present invention further relates to polynucleotides which hybridizeto the hereinabove-described sequences if there is at least 70%,preferably at least 90%, and more preferably at least 95% identitybetween the sequences. The present invention particularly relates topolynucleotides which hybridize under stringent conditions to thehereinabove-described polynucleotides. As herein used, the term"stringent conditions" means hybridization will occur only if there isat least 95% and preferably at least 97% identity between the sequences.The polynucleotides which hybridize to the hereinabove describedpolynucleotides in a preferred embodiment encode polypeptides whicheither retain substantially the same biological function or activity asthe mature polypeptide encoded by the cDNAs of FIG. 1(A-E) (SEQ ID NO:1)or the deposited cDNA(s).

Alternatively, the polynucleotide may have at least 20 bases, preferably30 bases, and more preferably at least 50 bases which hybridize to apolynucleotide of the present invention and which has an identitythereto, as hereinabove described, and which may or may not retainactivity. For example, such polynucleotides may be employed as probesfor the polynucleotide of SEQ ID NO:1, for example, for recovery of thepolynucleotide or as a diagnostic probe or as a PCR primer.

Thus, the present invention is directed to polynucleotides having atleast a 70% identity, preferably at least 90% and more preferably atleast a 95% identity to a polynucleotide which encodes the polypeptideof SEQ ID NO:2 as well as fragments thereof, which fragments have atleast 30 bases and preferably at least 50 bases and to polypeptidesencoded by such polynucleotides.

The deposit(s) referred to herein will be maintained under the terms ofthe Budapest Treaty on the International Recognition of the Deposit ofMicro-organisms for purposes of Patent Procedure. These deposits areprovided merely as convenience to those of skill in the art and are notan admission that a deposit is required under 35 U.S.C. §112. Thesequence of the polynucleotides contained in the deposited materials, aswell as the amino acid sequence of the polypeptides encoded thereby, areincorporated herein by reference and are controlling in the event of anyconflict with any description of sequences herein. A license may berequired to make, use or sell the deposited materials, and no suchlicense is hereby granted.

The present invention further relates to a VIGF polypeptide which hasthe deduced amino acid sequence of FIG. 1(A-E) (SEQ ID NO:2) or whichhas the amino acid sequence encoded by the deposited cDNA, as well asfragments, analogs and derivatives of such polypeptide.

The terms "fragment," "derivative" and "analog" when referring to thepolypeptide of FIG. 1 (SEQ ID NO:2) or that encoded by the depositedcDNA, means a polypeptide which retains essentially the same biologicalfunction or activity as such polypeptide. Thus, an analog includes aproprotein which can be activated by cleavage of the proprotein portionto produce an active mature polypeptide.

The polypeptide of the present invention may be a recombinantpolypeptide, a natural polypeptide or a synthetic polypeptide,preferably a recombinant polypeptide.

The fragment, derivative or analog of the polypeptide of FIG. 1(A-E)(SEQ ID NO:2) or that encoded by the deposited cDNA may be (i) one inwhich one or more of the amino acid residues are substituted with aconserved or non-conserved amino acid residue (preferably a conservedamino acid residue) and such substituted amino acid residue may or maynot be one encoded by the genetic code, or (ii) one in which one or moreof the amino acid residues includes a substituent group, or (iii) one inwhich the mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol), or (iv) one in which the additional amino acidsare fused to the mature polypeptide, such as a leader or secretorysequence or a sequence which is employed for purification of the maturepolypeptide or a proprotein sequence. Such fragments, derivatives andanalogs are deemed to be within the scope of those skilled in the artfrom the teachings herein.

The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

The term "isolated" means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

The polypeptides of the present invention include the polypeptide of SEQID NO:2 (in particular the mature polypeptide) as well as polypeptideswhich have at least 70% similarity (preferably at least 70% identity) tothe polypeptide of SEQ ID NO:2 and more preferably at least 90%similarity (more preferably at least 90% identity) to the polypeptide ofSEQ ID NO:2 and still more preferably at least 95% similarity (stillmore preferably at least 95% identity) to the polypeptide of SEQ ID NO:2and also include portions of such polypeptides with such portion of thepolypeptide generally containing at least 30 amino acids and morepreferably at least 50 amino acids.

As known in the art "similarity" between two polypeptides is determinedby comparing the amino acid sequence and its conserved amino acidsubstitutes of one polypeptide to the sequence of a second polypeptide.

Fragments or portions of the polypeptides of the present invention maybe employed for producing the corresponding full-length polypeptide bypeptide synthesis; therefore, the fragments may be employed asintermediates for producing the full-length polypeptides. Fragments orportions of the polynucleotides of the present invention may be used tosynthesize full-length polynucleotides of the present invention.

The present invention also relates to vectors which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques.

Host cells are genetically engineered (transduced or transformed ortransfected) with the vectors of this invention which may be, forexample, a cloning vector or an expression vector. The vector may be,for example, in the form of a plasmid, a viral particle, a phage, etc.The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying the VIGF genes. The culture conditions, suchas temperature, pH and the like, are those previously used with the hostcell selected for expression, and will be apparent to the ordinarilyskilled artisan.

The polynucleotides of the present invention may be employed forproducing polypeptides by recombinant techniques. Thus, for example, thepolynucleotide may be included in any one of a variety of expressionvectors for expressing a polypeptide. Such vectors include chromosomal,nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40;bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectorsderived from combinations of plasmids and phage DNA, viral DNA such asvaccinia, adenovirus, fowl pox virus, and pseudorabies. However, anyother vector may be used as long as it is replicable and viable in thehost.

The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site(s) by procedures known in theart. Such procedures and others are deemed to be within the scope ofthose skilled in the art.

The DNA sequence in the expression vector is operatively linked to anappropriate expression control sequence(s) (promoter) to direct mRNAsynthesis. As representative examples of such promoters, there may bementioned: LTR or SV40 promoter, the E. coli. lac or trp, the phagelambda P_(L) promoter and other promoters known to control expression ofgenes in prokaryotic or eukaryotic cells or their viruses. Theexpression vector also contains a ribosome binding site for translationinitiation and a transcription terminator. The vector may also includeappropriate sequences for amplifying expression.

In addition, the expression vectors preferably contain one or moreselectable marker genes to provide a phenotypic trait for selection oftransformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

The vector containing the appropriate DNA sequence as hereinabovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transform an appropriate host to permit the host toexpress the protein.

As representative examples of appropriate hosts, there may be mentioned:bacterial cells, such as E. coli, Streptomyces, Salmonella typhimurium;fungal cells, such as yeast; insect cells such as Drosophila S2 andSpodoptera Sf9; adenoviruses; animal cells such as CHO, COS or Bowesmelanoma; adenoviruses; plant cells, etc. The selection of anappropriate host is deemed to be within the scope of those skilled inthe art from the teachings herein.

More particularly, the present invention also includes recombinantconstructs comprising one or more of the sequences as broadly describedabove. The constructs comprise a vector, such as a plasmid or viralvector, into which a sequence of the invention has been inserted, in aforward or reverse orientation. In a preferred aspect of thisembodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially available. The following vectorsare provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen),pBS, pD10, phagescript, psiX174, pbluescript SK, pBSKS, pNH8A, pNH16a,pNH18A, pNH46A (Stratagene); pTRC99a, pKK223-3, pKK233-3, pDR540, pRIT5(Pharmacia). Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene)pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid orvector may be used as long as they are replicable and viable in thehost.

Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are PKK232-8 and PCM7. Particular namedbacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P_(R), P_(L)and trp. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art.

In a further embodiment, the present invention relates to host cellscontaining the above-described constructs. The host cell can be a highereukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell,such as a yeast cell, or the host cell can be a prokaryotic cell, suchas a bacterial cell. Introduction of the construct into the host cellcan be effected by calcium phosphate transfection, DEAE-Dextran mediatedtransfection, or electroporation (Davis, L., Dibner, M., Battey, I.,Basic Methods in Molecular Biology, (1986)).

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

Mature proteins can be expressed in mammalian cells, yeast, bacteria, orother cells under the control of appropriate promoters. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the present invention.Appropriate cloning and expression vectors for use with prokaryotic andeukaryotic hosts are described by Sambrook, et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), thedisclosure of which is hereby incorporated by reference.

Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes is increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act on a promoter to increase itstranscription. Examples including the SV40 enhancer on the late side ofthe replication origin bp 100 to 270, a cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), α-factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein into the periplasmic space orextracellular medium. Optionally, the heterologous sequence can encode afusion protein including an N-terminal identification peptide impartingdesired characteristics, e.g., stabilization or simplified purificationof expressed recombinant product.

Useful expression vectors for bacterial use are constructed by insertinga structural DNA sequence encoding a desired protein together withsuitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

As a representative but nonlimiting example, useful expression vectorsfor bacterial use can comprise a selectable marker and bacterial originof replication derived from commercially available plasmids comprisinggenetic elements of the well known cloning vector pBR322 (ATCC 37017).Such commercial vectors include, for example, pKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis.,USA). These pBR322 "backbone" sections are combined with an appropriatepromoter and the structural sequence to be expressed.

Following transformation of a suitable host strain and growth of thehost strain to an appropriate cell density, the selected promoter isinduced by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period.

Cells are typically harvested by centrifugation, disrupted by physicalor chemical means, and the resulting crude extract retained for furtherpurification.

Microbial cells employed in expression of proteins can be disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents, such methods arewell know to those skilled in the art.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts, described by Gluzman,Cell, 23:175 (1981), and other cell lines capable of expressing acompatible vector, for example, the C127, 3T3, CHO, HeLa and BHK celllines. Mammalian expression vectors will comprise an origin ofreplication, a suitable promoter and enhancer, and also any necessaryribosome binding sites, polyadenylation site, splice donor and acceptorsites, transcriptional termination sequences, and 5' flankingnontranscribed sequences. DNA sequences derived from the SV40 splice,and polyadenylation sites may be used to provide the requirednontranscribed genetic elements.

The VIGF polypeptides can be recovered and purified from recombinantcell cultures by methods including ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. Protein refolding steps can be used, as necessary, incompleting configuration of the mature protein. Finally, highperformance liquid chromatography (HPLC) can be employed for finalpurification steps.

The polypeptides of the present invention may be a naturally purifiedproduct, or a product of chemical synthetic procedures, or produced byrecombinant techniques from a prokaryotic or eukaryotic host (forexample, by bacterial, yeast, higher plant, insect and mammalian cellsin culture). Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. Polypeptides of the inventionmay also include an initial methionine amino acid residue.

This VIGF polypeptide of the present invention may be employed inwound-healing and associated therapies concerned with re-growth oftissue, such as connective tissue, skin, bone, cartilage, muscle, lungor kidney.

VIGF polypeptide may also be employed to enhance the growth of vascularsmooth muscle and endothelial cells leading to the stimulation ofangiogenesis. The VIGF-mediated increase in angiogenesis would bebeneficial to ischemic tissues and to collateral coronary development inthe heart subsequent to coronary stenosis.

VIGF polypeptide may also be employed during implant fixation tostimulate the growth of cells around the implant and therefore,facilitate its attachment to its intended site.

VIGF polypeptide may also be employed to increase IGF stability intissues or in serum. It may also increase binding to the IGF receptor.Since IGF has been shown in vitro to enhance human marrow erythroid andgranulocytic progenitor cell growth, VIGF polypeptide may also beemployed to stimulate erythropoiesis or granulopoiesis.

In accordance with yet a further aspect of the present invention, thereis provided a process for utilizing such polypeptides, orpolynucleotides encoding such polypeptides, as a research reagent for invitro purposes related to scientific research, synthesis of DNA andmanufacture of DNA vectors, for the purpose of developing therapeuticsand diagnostics for the treatment of human disease.

This invention provides a method for identification of the receptor forVIGF. The gene encoding the receptor can be identified by numerousmethods known to those of skill in the art, for example, ligand panningand FACS sorting (Coligan, et al., Current Protocols in Immun., 1(2),Chapter 5, (1991)). Preferably, expression cloning is employed whereinpolyadenylated RNA is prepared from a cell responsive to VIGF, and acDNA library created from this RNA is divided into pools and used totransfect COS cells or other cells that are not responsive to VIGF.Transfected cells which are grown on glass slides are exposed to labeledVIGF. VIGF can be labeled by a variety of means including iodination orinclusion of a recognition site for a site-specific protein kinase.Following fixation and incubation, the slides are subjected toautoradiographic analysis. Positive pools are identified and sub-poolsare prepared and retransfected using an iterative sub-pooling andrescreening process, eventually yielding a single clone that encodes theputative receptor.

As an alternative approach for receptor identification, labeled VIGF canbe photoaffinity linked with cell membrane or extract preparations thatexpress the receptor molecule. Cross-linked material is resolved by PAGEand exposed to X-ray film. The labeled complex containing theVIGF-receptor can be excised, resolved into peptide fragments, andsubjected to protein microsequencing. The amino acid sequence obtainedfrom microsequencing would be used to design a set of degenerateoligonucleotide probes to screen a cDNA library to identify the geneencoding the putative receptor.

This invention is also related to a method of screening compounds toidentify those which mimic VIGF (agonists) or prevent the effect ofVIGF. An example of such a method takes advantage of the ability of VIGFto stimulate the proliferation of endothelial cells in the presence ofthe comitogen Con A. Human umbilical vein endothelial cells are obtainedand cultured in 96-well flat-bottomed culture plates (Costar, Cambridge,Mass.) and supplemented with a reaction mixture appropriate forfacilitating proliferation of the cells, the mixture containing Con-A(Calbiochem, La Jolla, Calif.). Con-A and the compound to be screenedare added and after incubation at 37° C., cultures are pulsed with ³H!thymidine and harvested onto glass fiber filters (PhD; CambridgeTechnology, Watertown, Mass.). Mean ³ H!-thymidine incorporation (cpm)of triplicate cultures is determined using a liquid scintillationcounter (Beckman Instruments, Irvine, Calif.). Significant ³H!-thymidine incorporation indicates stimulation of endothelial cellproliferation.

To assay for antagonists, the assay described above is performed,however, in this assay VIGF is added along with the compound to bescreened and the ability of the compound to inhibit ³ H!-thymidineincorporation in the presence of VIGF, indicates that the compund is anantagonist to VIGF. Alternatively, VIGF antagonists may be detected bycombining VIGF and a potential antagonist with membrane-bound VIGFreceptors or recombinant receptors under appropriate conditions for acompetitive inhibition assay. VIGF can be labeled, such as byradioactivity, such that the number of VIGF molecules bound to thereceptor can determine the effectiveness of the potential antagonist.

Also, a mammalian cell or membrane preparation expressing the VIGFreceptor would be incubated with labeled VIGF in the presence of thecompound. The ability of the compound to enhance or block thisinteraction could then be measured. ALternatively, VIGF, labelled IGFand a potential compound could be incubated under conditions where VIGFwould naturally bind to IGF. The extent of this interaction could bemeasured to determine if the compound is an effective antagonist oragonist.

Examples of potential VIGF antagonists include an antibody, or in somecases, an oligonucleotide, which binds to the polypeptide.Alternatively, a potential antagonist may be a closely related protein,for example, a mutated form of VIGF, which recognizes the VIGF receptorbut imparts no effect, thereby competitively inhibiting the action ofVIGF.

Another potential VIGF antagonist is an antisense construct preparedusing antisense technology. Antisense technology can be used to controlgene expression through triple-helix formation or antisense DNA or RNA,both of which methods are based on binding of a polynucleotide to DNA orRNA. For example, the 5' coding portion of the polynucleotide sequence,which encodes for the mature polypeptides of the present invention, isused to design an antisense RNA oligonucleotide of from about 10 to 40base pairs in length. A DNA oligonucleotide is designed to becomplementary to a region of the gene involved in transcription (triplehelix -see Lee et al., Nucl. Acids Res., 6:3073 (1979); Cooney et al,Science, 241:456 (1988); and Dervan et al., Science, 251: 1360 (1991)),thereby preventing transcription and the production of VIGF. Theantisense RNA oligonucleotide hybridizes to the mRNA in vivo and blockstranslation of the mRNA molecule into the VIGF (antisense--Okano, J.Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitorsof Gene Expression, CRC Press, Boca Raton, Fla. (1988)). Theoligonucleotides described above can also be delivered to cells suchthat the antisense RNA or DNA may be expressed in vivo to inhibitproduction of VIGF.

Potential VIGF antagonists include small molecules which bind to theactive site, the receptor binding site, IGF or other growth factorbinding site of the polypeptide thereby blocking the normal biologicalactivity of VIGF. Examples of small molecules include but are notlimited to small peptides or peptide-like molecules.

The antagonists may be employed to inhibit tumor neovascularization andthe neointimal proliferation of smooth muscle cells prevalent inatherosclerosis and restenosis subsequent to balloon angioplasty.

The antagonists may also be employed to inhibit the over production ofscar tissue seen in a keloid which forms after surgery, fibrosis aftermyocardial infarction, or fibrotic lesions associated with pulmonaryfibrosis. The antagonists may be employed in a composition with apharmaceutically acceptable carrier, e.g., as hereinafter described.

The VIGF polypeptides and antagonist or agonists of the presentinvention may be employed in combination with a suitable pharmaceuticalcarrier. Such compositions comprise a therapeutically effective amountof the polypeptide, and a pharmaceutically acceptable carrier orexcipient. Such a carrier includes but is not limited to saline,buffered saline, dextrose, water, glycerol, ethanol, and combinationsthereof. The formulation should suit the mode of administration.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. In addition, thepharmaceutical compositions may be employed in conjunction with othertherapeutic compounds.

The pharmaceutical compositions may be administered in a convenientmanner such as by the oral, topical, intravenous, intraperitoneal,intramuscular, subcutaneous, intranasal or intradermal routes. Thepharmaceutical compositions are administered in an amount which iseffective for treating and/or prophylaxis of the specific indication. Ingeneral, they are administered in an amount of at least about 10 μg/kgbody weight and in most cases they will be administered in an amount notin excess of about 8 mg/Kg body weight per day. In most cases, thedosage is from about 10 μg/kg to about 1 mg/kg body weight daily, takinginto account the routes of administration, symptoms, etc.

VIGF in combination with other growth factors including but not limitedto, PDGF, IGF, FGF, EGF or TGF-β may accelerate physiological responsesas seen in wound healing.

The VIGF polypeptide and agonists and antagonists which arepolypeptides, may also be employed in accordance with the presentinvention by expression of such polypeptides in vivo, which is oftenreferred to as "gene therapy."

Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with theengineered cells then being provided to a patient to be treated with thepolypeptide. Such methods are well-known in the art. For example, cellsmay be engineered by procedures known in the art by use of a retroviralparticle containing RNA encoding a polypeptide of the present invention.

Similarly, cells may be engineered in vivo for expression of apolypeptide in vivo by, for example, procedures known in the art. Asknown in the art, a producer cell for producing a retroviral particlecontaining RNA encoding the polypeptide of the present invention may beadministered to a patient for engineering cells in vivo and expressionof the polypeptide in vivo. These and other methods for administering apolypeptide of the present invention by such method should be apparentto those skilled in the art from the teachings of the present invention.For example, the expression vehicle for engineering cells may be otherthan a retrovirus, for example, an adenovirus which may be used toengineer cells in vivo after combination with a suitable deliveryvehicle.

Retroviruses from which the retroviral plasmid vectors hereinabovementioned may be derived include, but are not limited to, Moloney MurineLeukemia Virus, spleen necrosis virus, retroviruses such as Rous SarcomaVirus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemiavirus, human immunodeficiency virus, adenovirus, MyeloproliferativeSarcoma Virus, and mammary tumor virus. In one embodiment, theretroviral plasmid vector is derived from Moloney Murine Leukemia Virus.

The vector includes one or more promoters. Suitable promoters which maybe employed include, but are not limited to, the retroviral LTR; theSV40 promoter; and the human cytomegalovirus (CMV) promoter described inMiller, et al., Biotechniques, Vol. 7, No. 9, 980-990 (1989), or anyother promoter (e.g., cellular promoters such as eukaryotic cellularpromoters including, but not limited to, the histone, pol III, andβ-actin promoters). Other viral promoters which may be employed include,but are not limited to, adenovirus promoters, thymidine kinase (TK)promoters, and B19 parvovirus promoters. The selection of a suitablepromoter will be apparent to those skilled in the art from the teachingscontained herein.

The nucleic acid sequence encoding the polypeptide of the presentinvention is under the control of a suitable promoter. Suitablepromoters which may be employed include, but are not limited to,adenoviral promoters, such as the adenoviral major late promoter; orhetorologous promoters, such as the cytomegalovirus (CMV) promoter; therespiratory syncytial virus (RSV) promoter; inducible promoters, such asthe MMT promoter, the metallothionein promoter; heat shock promoters;the albumin promoter; the ApoAI promoter; human globin promoters; viralthymidine kinase promoters, such as the Herpes Simplex thymidine kinasepromoter; retroviral LTRs (including the modified retroviral LTRshereinabove described); the β-actin promoter; and human growth hormonepromoters. The promoter also may be the native promoter which controlsthe gene encoding the polypeptide.

The retroviral plasmid vector is employed to transduce packaging celllines to form producer cell lines. Examples of packaging cells which maybe transfected include, but are not limited to, the PE501, PA317, ψ-2,ψ-AM, PA12, T19-14X, VT-19-17-H2, ψCRE, ψCRIP, GP+E-86, GP+envAm12, andDAN cell lines as described in Miller, Human Gene Therapy, Vol. 1, pgs.5-14 (1990), which is incorporated herein by reference in its entirety.The vector may transduce the packaging cells through any means known inthe art. Such means include, but are not limited to, electroporation,the use of liposomes, and CaPO₄ precipitation. In one alternative, theretroviral plasmid vector may be encapsulated into a liposome, orcoupled to a lipid, and then administered to a host.

The producer cell line generates infectious retroviral vector particleswhich include the nucleic acid sequence(s) encoding the polypeptides.Such retroviral vector particles then may be employed, to transduceeukaryotic cells, either in vitro or in vivo. The transduced eukaryoticcells will express the nucleic acid sequence(s) encoding thepolypeptide. Eukaryotic cells which may be transduced include, but arenot limited to, embryonic stem cells, embryonic carcinoma cells, as wellas hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts,keratinocytes, endothelial cells, and bronchial epithelial cells.

This invention is also related to the use of the VIGF gene as adiagnostic. Detection of a mutated form of VIGF will allow a diagnosisof a disease or a susceptibility to a disease, such as a tumor, sincemutations in VIGF may cause tumors.

Individuals carrying mutations in the human VIGF gene may be detected atthe DNA level by a variety of techniques. Nucleic acids for diagnosismay be obtained from a patient's cells, such as from blood, urine,saliva, tissue biopsy and autopsy material. The genomic DNA may be useddirectly for detection or may be amplified enzymatically by using PCR(Saiki et al., Nature, 324:163-166 (1986)) prior to analysis. RNA orcDNA may also be used for the same purpose. As an example, PCR primerscomplementary to the nucleic acid encoding VIGF can be used to identifyand analyze VIGF mutations. For example, deletions and insertions can bedetected by a change in size of the amplified product in comparison tothe normal genotype. Point mutations can be identified by hybridizingamplified DNA to radiolabeled VIGF RNA or alternatively, radiolabeledVIGF antisense DNA sequences. Perfectly matched sequences can bedistinguished from mismatched duplexes by RNase A digestion or bydifferences in melting temperatures.

Genetic testing based on DNA sequence differences may be achieved bydetection of alteration in electrophoretic mobility of DNA fragments ingels with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamidine gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g., Myerset al., Science, 230:1242 (1985)).

Sequence changes at specific locations may also be revealed by nucleaseprotection assays, such as RNase and S1 protection or the chemicalcleavage method (e.g., Cotton et al., PNAS, USA, 85:4397-4401 (1985)).

Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, (e.g.,Restriction Fragment Length Polymorphisms (RFLP)) and Southern blottingof genomic DNA.

In addition to more conventional gel-electrophoresis and DNA sequencing,mutations can also be detected by in situ analysis.

VIGF protein expression may be linked to vascular disease orneovascularization associated with tumor formation. VIGF has a signalpeptide and the mRNA is highly expressed in endothelial cells and to alesser extent in smooth muscle cells which indicates that the protein ispresent in serum. Accordingly, an anti-VIGF antibody could be used todiagnose vascular disease or neovascularization associated with tumorformation since an altered level of this polypeptide may be indicativeof such disorders.

A competition assay may be employed wherein antibodies specific to VIGFis attached to a solid support and labeled VIGF and a sample derivedfrom the host are passed over the solid support and the amount of labeldetected attached to the solid support can be correlated to a quantityof VIGF in the sample.

The sequences of the present invention are also valuable for chromosomeidentification. The sequence is specifically targeted to and canhybridize with a particular location on an individual human chromosome.Moreover, there is a current need for identifying particular sites onthe chromosome. Few chromosome marking reagents based on actual sequencedata (repeat polymorphisms) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with genes associated with disease.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers(preferably 15-25 bp) from the cDNA. Computer analysis of the 3'untranslated region is used to rapidly select primers that do not spanmore than one exon in the genomic DNA, thus complicating theamplification process. These primers are then used for PCR screening ofsomatic cell hybrids containing individual human chromosomes. Only thosehybrids containing the human gene corresponding to the primer will yieldan amplified fragment.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular DNA to a particular chromosome. Using the present inventionwith the same oligonucleotide primers, sublocalization can be achievedwith panels of fragments from specific chromosomes or pools of largegenomic clones in an analogous manner. Other mapping strategies that cansimilarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphasechromosomal spread can be used to provide a precise chromosomal locationin one step. This technique can be used with cDNA as short as 50 or 60bases. For a review of this technique, see Verma et al., HumanChromosomes: a Manual of Basic Techniques, Pergamon Press, New York(1988).

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. Such data are found, for example, in V. McKusick,Mendelian Inheritance in Man (available on line through Johns HopkinsUniversity Welch Medical Library). The relationship between genes anddiseases that have been mapped to the same chromosomal region are thenidentified through linkage analysis (coinheritance of physicallyadjacent genes).

Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

The polypeptides, their fragments or other derivatives, or analogsthereof, or cells expressing them can be used as an immunogen to produceantibodies thereto. These antibodies can be, for example, polyclonal ormonoclonal antibodies. The present invention also includes chimeric,single chain, and humanized antibodies, as well as Fab fragments, or theproduct of an Fab expression library. Various procedures known in theart may be used for the production of such antibodies and fragments.

Antibodies generated against the polypeptides corresponding to asequence of the present invention can be obtained by direct injection ofthe polypeptides into an animal or by administering the polypeptides toan animal, preferably a nonhuman. The antibody so obtained will thenbind the polypeptides itself. In this manner, even a sequence encodingonly a fragment of the polypeptides can be used to generate antibodiesbinding the whole native polypeptides. Such antibodies can then be usedto isolate the polypeptide from tissue expressing that polypeptide.

For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique (Kohler and Milstein, 1975,Nature, 256:495-497), the trioma technique, the human B-cell hybridomatechnique (Kozbor et al., 1983, Immunology Today 4:72), and theEBV-hybridoma technique to produce human monoclonal antibodies (Cole, etal., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc., pp. 77-96).

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies toimmunogenic polypeptide products of this invention. Also, transgenicmice may be used to express humanized antibodies to immunogenicpolypeptide products of this invention.

The present invention will be further described with reference to thefollowing examples; however, it is to be understood that the presentinvention is not limited to such examples. All parts or amounts, unlessotherwise specified, are by weight.

In order to facilitate understanding of the following examples certainfrequently occurring methods and/or terms will be described.

"Plasmids" are designated by a lower case p preceded and/or followed bycapital letters and/or numbers. The starting plasmids herein are eithercommercially available, publicly available on an unrestricted basis, orcan be constructed from available plasmids in accord with publishedprocedures. In addition, equivalent plasmids to those described areknown in the art and will be apparent to the ordinarily skilled artisan.

"Digestion" of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion the reaction is electrophoreseddirectly on a polyacrylamide gel to isolate the desired fragment.

Size separation of the cleaved fragments is performed using 8 percentpolyacrylamide gel described by Goeddel, D. et al., Nucleic Acids Res.,8:4057 (1980).

"Oligonucleotides" refers to either a single strandedpolydeoxynucleotide or two complementary polydeoxynucleotide strandswhich may be chemically synthesized. Such synthetic oligonucleotideshave no 5' phosphate and thus will not ligate to another oligonucleotidewithout adding a phosphate with an ATP in the presence of a kinase. Asynthetic oligonucleotide will ligate to a fragment that has not beendephosphorylated.

"Ligation" refers to the process of forming phosphodiester bonds betweentwo double stranded nucleic acid fragments (Maniatis, T., et al., Id.,p. 146). Unless otherwise provided, ligation may be accomplished usingknown buffers and conditions with 10 units of T4 DNA ligase ("ligase")per 0.5 μg of approximately equimolar amounts of the DNA fragments to beligated.

Unless otherwise stated, transformation was performed as described inthe method of Graham, F. and Van der Eb, A., Virology, 52:456-457(1973).

EXAMPLE 1

Bacterial Expression and Purification of VIGF

The DNA sequence encoding VIGF, ATCC #75874, was initially amplifiedusing PCR oligonucleotide primers corresponding to the 5' sequences ofthe processed VIGF protein (minus the signal peptide sequence) and thevector sequences 3' to the VIGF gene. Additional nucleotidescorresponding to VIGF were added to the 5' and 3' sequencesrespectively. The 5' oligonucleotide primer has the sequence 5'CGCAAGCTTAAATAATTATGCGGTGGACTGC 3' (SEQ ID NO:3) contains a Hind IIIrestriction enzyme site (in bold) followed by 21 nucleotides of VIGFcoding sequence starting from the presumed terminal amino acid of theprocessed protein codon (underlined). The 3' oligonucleotide primer 5'CGCTCTAGATCAGCGTGGATTTAACCA 3' (SEQ ID NO:4) contains an Xba Irestriction site (in bold) followed by the reverse complement ofnucleotides corresponding to the carboxy-terminal 5 amino acids and thetranslational stop codon (underlined). The restriction enzyme sitescorrespond to the restriction enzyme sites on the bacterial expressionvector pQE-9 (Qiagen, Inc. Chatsworth, Calif.,). pQE-9 encodesantibiotic resistance (Amp^(r)), a bacterial origin of replication(ori), an IPTG-regulatable promoter operator (P/O), a ribosome bindingsite (RBS), a 6-His tag and restriction enzyme sites. The VIGF PCRproduct and pQE-9 were then digested with Hind III and Xba I and ligatedtogether with T4 DNA ligase. The desired recombinants would contain theVIGF coding sequence inserted downstream from the pQE-9 encodedhistidine tag and the ribosome binding site. The ligation mixture wasthen used to transform E. coli strain M15 pREP4! (Qiagen, Inc.) by theprocedure described in Sambrook, J. et al., Molecular Cloning: ALaboratory Manual, Cold Spring Laboratory Press, (1989). M15 pREP4!contains multiple copies of the plasmid pREP4, which expresses the lacIrepressor and also confers kanamycin resistance (Kan^(r)). Transformantswere identified by their ability to grow on LB plates andampicillin/kanamycin resistant colonies were selected. Plasmid DNA wasisolated and confirmed by restriction analysis. Clones containing thedesired constructs were grown overnight (O/N) in liquid culture in LBmedia supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml). The O/Nculture was used to inoculate a large culture at a ratio of 1:100 to1:250. The cells were grown to an optical density 600 (O.D.⁶⁰⁰) ofbetween 0.4 and 0.6. IPTG ("Isopropyl-B-D-thiogalacto pyranoside") wasthen added to a final concentration of 1 mM. IPTG induces byinactivating the lacI repressor, clearing the P/O leading to increasedgene expression. Cells were grown an extra 3 to 4 hours such that thereis an exponential growth culture present. Cells were then harvested bycentrifugation. The VIGF/6-Histidine-containing M15 pREP4! cells werelysed in 6M GnHCl, 50 mM NaPO₄ at pH 8.0. The lysate was loaded on aNickel-Chelate column and the flow-through collected. The column waswashed with 6M GnHCl, 50 mM NaPO₄ at pH 8.0, 6.0 and 5.0. The VIGFfusion protein (>90% pure) was eluted at pH 2.0. For the purpose ofrenaturation, the pH 2.0 eluate was adjusted to 3 molar guanidine HCl,100 mM sodium phosphate, 10 mmolar glutathione (reduced) and 2 mmolarglutathione (oxidized). After incubation in this solution for 12 hoursthe protein was dialyzed to 10 mmolar sodium phosphate. To run the gel,the pellets were resuspended in SDS/NaOH and SDS-PAGE loading buffer,heat denatured, then electrophoresed on a 15% denaturing polyacrylamidegel. The Gibco BRL low range molecular weight standard was alsoelectrophoresed (lane 1). The proteins were visualized with CoomassieBrilliant Blue R-250 stain.

Example 2

Cloning and Expression of VIGF Using the Baculovirus Expression System

The DNA sequence encoding the full length VIGF protein, ATCC #75874, isdigested with the restriction enzymes PvuII and XbaI. The 639 nucleotidePvuII, XbaI fragment contains the entire VIGF coding region plus 11 and77 nucleotides of 5' and 3' untranslated DNA, respectively. Thisfragment, designated F2, is isolated from a 1% agarose gel using acommercially available kit ("Geneclean", BIO 101 Inc., La Jolla,Calif.).

The vector pA2 is used for the expression of the VIGF protein using thebaculovirus expression system (for review see: Summers, M. D. and Smith,G. E. 1987, A manual of methods for baculovirus vectors and insect cellculture procedures, Texas Agricultural Experimental Station Bulletin No.1555). This expression vector contains the strong polyhedrin promoter ofthe Autographa californica nuclear polyhidrosis virus (AcMNPV) followedby the recognition sites for the restriction endonucleases SmaI andXbaI. The polyadenylation site of the simian virus (SV)40 is used forefficient polyadenylation. For an easy selection of recombinant virusesthe beta-galactosidase gene from E.coli is inserted in the sameorientation as the polyhedrin promoter followed by the polyadenylationsignal of the polyhedrin gene. The polyhedrin sequences are flanked atboth sides by viral sequences for the cell-mediated homologousrecombination of cotransfected wild-type viral DNA. Many otherbaculovirus vectors could be used in place of pA2 such as, PRG1, pAc373,pVL941 and pAcIM1 (Luckow, V. A. and Summers, M. D., Virology,170:31-39).

The plasmid is digested with the restriction enzymes SmaI and XbaI andthen dephosphorylated using calf intestinal phosphatase by proceduresknown in the art. The DNA is then isolated from a 1% agarose gel usingthe commercially available kit ("Geneclean" BIO 101 Inc., La Jolla,Calif.). This vector DNA is designated V2.

Fragment F2 and the dephosphorylated plasmid V2 are ligated with T4 DNAligase. E.coli strain XL1 Blue (Stratagene Cloning Systems, 11011 NorthTorrey Pines Road La Jolla, Calif. 92037) are then transformed andbacteria identified that contained the plasmid (pBac VIGF) with the VIGFcDNA using the enzymes BamHI and XbaI. The sequence of the clonedfragment is confirmed by DNA sequencing.

5 μg of the plasmid pBac VIGF is cotransfected with 1.0 μg of acommercially available linearized baculovirus ("BaculoGold™ baculovirusDNA", Pharmingen, San Diego, Calif.) using the lipofection method(Felgner et al. Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)).

1 μg of BaculoGold™ virus DNA and 5 μg of the plasmid pBac VIGF aremixed in a sterile well of a microtiter plate containing 50 μl of serumfree Grace's medium (Life Technologies Inc., Gaithersburg, Md.).Afterwards 10 μl Lipofectin plus 90 μl Grace's medium are added, mixedand incubated for 15 minutes at room temperature. Then the transfectionmixture is added dropwise to the Sf9 insect cells (ATCC CRL 1711) seededin a 35 mm tissue culture plate with 1 ml Grace' medium without serum.The plate is rocked back and forth to mix the newly added solution. Theplate is then incubated for 5 hours at 27° C. After 5 hours thetransfection solution is removed from the plate and 1 ml of Grace'sinsect medium supplemented with 10% fetal calf serum is added. The plateis put back into an incubator and cultivation continued at 27° C. forfour days.

After four days the supernatant is collected and a plaque assayperformed similar as described by Summers and Smith (supra). As amodification an agarose gel with "Blue Gal" (Life Technologies Inc.,Gaithersburg) is used which allows an easy isolation of blue stainedplaques. (A detailed description of a "plaque assay" can also be foundin the user's guide for insect cell culture and baculovirologydistributed by Life Technologies Inc., Gaithersburg, page 9-10).

Four days after the serial dilution, the viruses are added to the cellsand blue stained plaques are picked with the tip of an Eppendorfpipette. The agar containing the recombinant viruses is then resuspendedin an Eppendorf tube containing 200 μl of Grace's medium. The agar isremoved by a brief centrifugation and the supernatant containing therecombinant baculoviruses is used to infect Sf9 cells seeded in 35 mmdishes. Four days later the supernatants of these culture dishes areharvested and then stored at 4° C.

Sf9 cells are grown in Grace's medium supplemented with 10%heat-inactivated FBS. The cells are infected with the recombinantbaculovirus V-VIGF at a multiplicity of infection (MOI) of 2. Six hourslater the medium is removed and replaced with SF900 II medium minusmethionine and cysteine (Life Technologies Inc., Gaithersburg). 42 hourslater 5 μCi of ³⁵ S-methionine and 5 μCi ³⁵ S cysteine (Amersham) areadded. The cells are further incubated for 16 hours before they areharvested by centrifugation and the labelled proteins visualized bySDS-PAGE and autoradiography.

Example 3

Expression of Recombinant VIGF in CHO Cells

The vector pN346 is used for the expression of the VIGF protein. PlasmidpN346 is a derivative of the plasmid pSV2-dhfr ATCC Accession No.37146!. Both plasmids contain the mouse dhfr gene under control of theSV40 early promoter. Chinese hamster ovary or other cells lackingdihydrofolate activity that are transfected with these plasmids can beselected by growing the cells in a selective medium (alpha minus MEM,Lift Technologies) supplemented with the chemotherapeutic agentmethotrexate. The amplication of the DHFR genes in cells resistant tomethotrexate (MTX) has been well documented (see, e.g., Alt, F. W.,Kellems, R. M., Bertino, J. R., and Schimke, R. T., 1978, J. Biol. Chem.253:1357-1370, Hamlin, J. L. and Ma, C. 1990, Biochem. et Biophys. Acta,1097:107-143, Page, M. J. and Sydenham, M. A. 1991, Biotechnology Vol.9:64-68). Cells grown in increasing concentrations of MTX developresistance to the drug by overproducing the target enzyme, DHFR, as aresult of amplification of the DHFR gene. If a second gene is linked tothe dhfr gene it is usually co-amplified and overexpressed.Subsequently, when the methotrexate is withdrawn, cell lines contain theamplified gene integrated into the chromosome(s).

Plasmid pN346 contains for the expression of the gene of interest astrong promoter of the long terminal repeat (LTR) of the Rouse SarcomaVirus (Cullen, et al., Molecular and Cellular Biology, March 1985,438-447) plus a fragment isolated from the enhancer of the immediateearly gene of human cytomegalovirus (CMV) (Boshart et al., Cell41:521-530, 1985). Downstream of the promoter are the following singlerestriction enzyme cleavage sites that allow the integration of thegenes: BamHI, Pvull, and Nrul. Behind these cloning sites the plasmidcontains translational stop codons in all three reading frames followedby the 3' intron and the polyadenylation site of the rat preproinsulingene. Other high efficient promoters can also be used for theexpression, e.g., the human β-actin promoter, the SV40 early or latepromoters or the long terminal repeats from other retroviruses, e.g.,HIV and HTLVI. For the polyadenylation of the mRNA other signals, e.g.,from the human growth hormone or globin genes can be used as well.

Stable cell lines carrying a gene of interest integrated into thechromosome can also be selected upon co-transfection with a selectablemarker such as gpt, G418 or hygromycin. It is advantageous to use morethan one selectable marker in the beginning, e.g. G418 plusmethotrexate.

The plasmid pN346 is digested with the restriction enzyme BamHI and thendephosphorylated using calf intestinal phosphatase by procedures knownin the art. The vector is then isolated from a 1% agarose gel.

The DNA sequence encoding the full length VIGF protein, ATCC #75874, isamplified using PCR oligonucleotide primers corresponding to the 5' and3' sequences of the gene:

The 5' primer has the sequence 5' CGCAGATCTCCGCCACCATGAAGAGCGTCTTGCTGCTG 3' (SEQ ID NO:5) and contains a BglII restrictionenzyme site (in bold) followed by 8 nucleotides resembling an efficientsignal for the initiation of translation in eukaryotic cells (Kozak, M.,J. Mol. Biol., 196:947-950, (1987)). The remaining nucleotidescorrespond to the amino terminal 7 amino acids including thetranslational initiation codon (underlined). The 3' primer has thesequence 5' CGCAGATCTAGCCTTCTCTCAGAAATCACA 3' (SEQ ID NO:6) and containsa BglII restriction site (in bold) and 21 nucleotides that are thereverse complement of 3' untranslated DNA starting 7 nucleotidesdownstream from the translational stop codon. The PCR product isdigested with BglII and purified on a 1% agarose gel using acommercially available kit ("Geneclean," BIO 101 Inc., La Jolla,Calif.). This fragment is then ligated to BamHI digested, phosphatasedpN346 plasmid with T4 DNA ligase. Xl1Blue (Stratagene) E. coli aretransformed and plated on LB, 50 μg/ml ampicillin plates. Coloniesbearing the desired recombinant in the proper orientation are screenedfor by PCR with a 5' primer which corresponds to the Rous sarcoma viruspromoter and a 3' primer which corresponds to the reverse complement ofVIGF codons 73-79. The sequence of the cloned fragment is confirmed byDNA sequencing. Transfection of CHO-dhfr-cells

Chinese hamster ovary cells lacking an active DHFR enzyme are used fortransfection. 5 μg of the expression plasmid pN346VIGF are cotransfectedwith 0.5 μg of the plasmid pSVneo using the lipofectin method (Felgneret al., supra). The plasmid pSV2-neo contains a dominant selectablemarker, the gene neo from Tn5 encoding an enzyme that confers resistanceto a group of antibiotics including G418. The cells are seeded in alphaminus MEM supplemented with 1 mg/ml G418. After 2 days, the cells aretrypsinized and seeded in hybridoma cloning plates (Greiner, Germany)and cultivated from 10-14 days. After this period, single clones aretrypsinized and then seeded in 6-well petri dishes using differentconcentrations of methotrexate (25, 50 nm, 100 nm, 200 nm, 400 nm).Clones growing at the highest concentrations of methotrexate are thentransferred to new 6-well plates containing even higher concentrationsof methotrexate (500 nM, 1 μM, 2 μM, 5 μM). The same procedure isrepeated until clones grew at a concentration of 100 μM.

The expression of the desired gene product is analyzed by Western blotanalysis and SDS-PAGE.

Example 4

Tissue Localization of VIGF Gene Expression by Northern Blot Analysis

A multiple tissue Northern blot (Clontech Laboratories, Inc., 4030Fabian Way; Palo Alto, Calif. 94303) containing 2 ug of human adultbrain, heart, placenta, lung, liver skeletal muscle, kidney, andpancreas poly A+ mRNA per lane is prehybridized in Church buffer(Church, G. M. & Gilbert, W., Proc. Natl. Acad. Sci. USA 81, 1991-1995(1984)) at 60° C. for one hour. The DNA sequence coding for VIGF,ATCC#75874, is amplified from the full length cDNA cloned in pBluescriptSK(-) using the M13 Forward (5' GGGTTTTCCCAGTCACGAC 3') (SEQ ID NO:7)and Reverse (5' ATGCTTCCGGCTCGTATG 3') (SEQ ID NO:8) primers.Twenty-five nanograms of PCR product is random primer radiolabeled(Prime-It II, Stratagene Cloning Systems, 11011 North Torrey Pines Rd.;La Jolla, Calif. 92037) with ³² P-dCTP. The heat denatured VIGF probe isadded directly to the prehybridization buffer and incubated 16 hr at 60°C. Two ten minute washes are performed in 0.2X SSC, 0.1% SDS at 60° C.Autoradiography is performed at -80° C.

A 2.3 kb transcript is seen in lung and kidney after a four dayexposure.

Example 5

Cell-Type Analysis of VIGF Gene Expression by Northern Blot Analysis

Human umbilical vein endothelial, aortic smooth muscle, dermal foreskinfibroblast cells (Clonetics, 9620 Chesapeake Drive, Suite #201; SanDiego, Calif. 92123) were grown to 75-90% confluency. Total RNA isextracted with RNAzol (Biotecx Laboratories, Inc., 6023 South Loop EastHouston, Tex. 77033). A 1.2% agarose formaldehyde gel is prepared andrun with 20 ug of total RNA per lane and an RNA ladder size marker (LifeTechnologies, Inc., 8400 Helgerman Ct., P.O. Box 6009 Gaithersburg, Md.20884) according to Sambrook et al. (1989). The RNA is transferredovernight to Hybond N+ (Amersham Corp., 2636 South Clearbrook Drive;Arlington Heights, Ill. 60005) and bound to the membrane with aStratalinker UV Crosslinker (Stratagene Cloning Systems, La Jolla,Calif.). The blot is prehybridized in Church buffer (Church, G. M. &Gilbert, W., PNAS, USA 81:1991-1995 (1984)) at 60° C. for one hour. TheDNA sequence encoding VIGF, ATCC #75874, is amplified from the fulllength cDNA cloned in pBluescript SK(-) using the M13 Forward (5'GGGTTTTCCCAGTCACGAC 3') (SEQ ID NO:9) and Reverse (5' ATGCTTCCGGCTCGTATG3') (SEQ ID NO:10) primers. Twenty-five nanograms of PCR product israndom primer radiolabeled (Prime-It II, Stratagene) with ³² P-dCTP. Theheat denatured VIGF probe is added directly to the prehybridizationbuffer and incubated 16 hr at 60° C. Two ten minute washes wereperformed in 0.2X SSC, 0.1% SDS at 60° C. Autoradiography is performedat -80° C. A 2.3-2.4 kb transcript is seen in umbilical vein endothelial(lane 1) and aortic smooth muscle cells (lane 2) after a two hourexposure and also in dermal foreskin fibroblast (lane 3) cells after a36 hour exposure.

Example 6

Expression Via Gene Therapy

Fibroblasts are obtained from a subject by skin biopsy. The resultingtissue is placed in tissue-culture medium and separated into smallpieces. Small chunks of the tissue are placed on a wet surface of atissue culture flask, approximately ten pieces are placed in each flask.The flask is turned upside down, closed tight and left at roomtemperature over night. After 24 hours at room temperature, the flask isinverted and the chunks of tissue remain fixed to the bottom of theflask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillinand streptomycin, is added. This is then incubated at 37° C. forapproximately one week. At this time, fresh media is added andsubsequently changed every several days. After an additional two weeksin culture, a monolayer of fibroblasts emerge. The monolayer istrypsinized and scaled into larger flasks.

pMV-7 (Kirschmeier, P. T. et al, DNA, 7:219-25 (1988) flanked by thelong terminal repeats of the Moloney murine sarcoma virus, is digestedwith EcoRI and HindIII and subsequently treated with calf intestinalphosphatase. The linear vector is fractionated on agarose gel andpurified, using glass beads.

The cDNA encoding a polypeptide of the present invention is amplifiedusing PCR primers which correspond to the 5' and 3' end sequencesrespectively. The 5' primer containing an EcoRI site and the 3' primer$further includes a HindIII site. Equal quantities of the Moloney murinesarcoma virus linear backbone and the amplified $EcoRI and HindIIIfragment are added together, in the presence of T4 DNA ligase. Theresulting mixture is maintained under conditions appropriate forligation of the two fragments. The ligation mixture is used to transformbacteria HB101, which are then plated onto agar-containing kanamycin forthe purpose of confirming that the vector had the gene of interestproperly inserted.

The amphotropic pA317 or GP+am12 packaging cells are grown in tissueculture to confluent density in Dulbecco's Modified Eagles Medium (DMEM)with 10% calf serum (CS), penicillin and streptomycin. The MSV vectorcontaining the gene is then added to the media and the packaging cellsare transduced with the vector. The packaging cells now produceinfectious viral particles containing the gene (the packaging cells arenow referred to as producer cells).

Fresh media is added to the transduced producer cells, and subsequently,the media is harvested from a 10 cm plate of confluent producer cells.The spent media, containing the infectious viral particles, is filteredthrough a millipore filter to remove detached producer cells and thismedia is then used to infect fibroblast cells. Media is removed from asub-confluent plate of fibroblasts and quickly replaced with the mediafrom the producer cells. This media is removed and replaced with freshmedia. If the titer of virus is high, then virtually all fibroblastswill be infected and no selection is required. If the titer is very low,then it is necessary to use a retroviral vector that has a selectablemarker, such as neo or his.

The engineered fibroblasts are then injected into the host, either aloneor after having been grown to confluence on cytodex 3 microcarrierbeads. The fibroblasts now produce the protein product.

Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, within thescope of the appended claims, the invention may be practiced otherwisethan as particularly described.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 17                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1271 BASE PAIRS                                                   (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       CTGCTTCCCACCAGCAAAGACCACGACTGGAGAGCCGAGCCGGAGCAGCTGGGAAACATG60                AAGAGCGTCTTGCTGCTGACCACGCTCCTCGTGCCTGCACACCTGGTGGCCGCCTGGAGC120               AATAATTATGCGGTGGACTGCCCTCAACACTGTGACAGCAGTGAGTGCAAAAGCAGCCCG180               CGCTGCAAGAGGACAGTGCTCGACGACTGTGGCTGCTGCCGAGTGTGCGCTGCAGGGCGG240               GGAGAAACTTGCTACCGCACAGTCTCAGGCATGGATGGCATGAAGTGTGGCCCGGGGCTG300               AGGTGTCAGCCTTCTAATGGGGAGGATCCTTTTGGTGAAGAGTTTGGTATCTGCAAAGAC360               TGTCCCTACGGCACCTTCGGGATGGATTGCAGAGAGACCTGCAACTGCCAGTCAGGCATC420               TGTGACAGGGGGACGGGAAAATGCCTGAAATTCCCCTTCTTCCAATATTCAGTAACCAAG480               TCTTCCAACAGATTTGTTTCTCTCACGGAGCATGACATGGCATCTGGAGATGGCAATATT540               GTGAGAGAAGAAGTTGTGAAAGAGAATGCTGCCGGGTCTCCCGTAATGAGGAAATGGTTA600               AATCCACGCTGATCCCGGCTGTGATTTCTGAGAGAAGGCTCTATTTTCGTGAYTGTTCAA660               CACACAGCCAACATTTTAGGAACTTTCTAGATTATAGCATAAGGACATGTAATTTTTGAA720               GACCAAATGTGATGCATGGTGGATCCAGAAAACAAAAAGTAGGATACTTACAATCCATAA780               CATCCATATGACTGAACACTTGTATGTGTTTGTTAAATATTCGAATGCATGTAGATTTGT840               TAAATGTGTGTGTATAGTAACACTGAAGAACTAAAAATGCAATTTAGGTAATCTTACATG900               GAGACAGGTCAACCAAAGAGGGAGCTAGGCAAAGCTGAAGACCGCAGTGAGTCAAATTAG960               TTCTTTGACTTTGATGTACATTAATGTTGGGATATGGAATGAAGACTTAAGAGCAGGAGA1020              AGATGGGGAGGGGGTGGGAGTGGGAAATAAAATATTTAGCCCTTCCTTGGTAGGTAGCTT1080              CTCTAGAATTTAATTRTGCTTTTTTTTTTTTTTTTGGGCTTTGGGAAAAGTCAAAATAAA1140              ACAACCAGAAAACCCCTGAAGGAAGTAAGATGTTTGAAGCTTATGGAAATTTGAGTAACA1200              AACAGCTTTGANCTGAGAGCAATTYCAAAAGGCTGCTGATGTAGCCCCCGGGTTNCCTNT1260              NTCTNAAGGAC1271                                                               (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 184 AMINO ACIDS                                                   (B) TYPE: AMINO ACID                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: PROTEIN                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetLysSerValLeuLeuLeuThrThrLeuLeuValProAlaHis                                 20-15-10                                                                      LeuValAlaAlaTrpSerAsnAsnTyrAlaValAspCysProGln                                 515                                                                           HisCysAspSerSerGluCysLysSerSerProArgCysLysArg                                 101520                                                                        ThrValLeuAspAspCysGlyCysCysArgValCysAlaAlaGly                                 253035                                                                        ArgGlyGluThrCysTyrArgThrValSerGlyMetAspGlyMet                                 404550                                                                        LysCysGlyProGlyLeuArgCysGlnProSerAsnGlyGluAsp                                 556065                                                                        ProPheGlyGluGluPheGlyIleCysLysAspCysProTyrGly                                 707580                                                                        ThrPheGlyMetAspCysArgGluThrCysAsnCysGlnSerGly                                 859095                                                                        IleCysAspArgGlyThrGlyLysCysLeuLysPheProPhePhe                                 100105110                                                                     GlnTyrSerValThrLysSerSerAsnArgPheValSerLeuThr                                 115120125                                                                     GluHisAspMetAlaSerGlyAspGlyAsnIleValArgGluGlu                                 130135140                                                                     ValValLysGluAsnAlaAlaGlySerProValMetArgLysTrp                                 145150155                                                                     LeuAsnProArg                                                                  160                                                                           (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 BASE PAIRS                                                     (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       CGCAAGCTTAAATAATTATGCGGTGGACTGC31                                             (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 BASE PAIRS                                                     (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       CGCTCTAGATCAGCGTGGATTTAACCA27                                                 (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 BASE PAIRS                                                     (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       CGCAGATCTCCGCCACCATGAAGAGCGTCTTGCTGCTG38                                      (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 BASE PAIRS                                                     (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       CGCAGATCTAGCCTTCTCTCAGAAATCACA30                                              (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 BASE PAIRS                                                     (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       GGGTTTTCCCAGTCACGAC19                                                         (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 BASE PAIRS                                                     (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       ATGCTTCCGGCTCGTATG18                                                          (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 BASE PAIRS                                                     (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       GGGTTTTCCCAGTCACGAC19                                                         (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 BASE PAIRS                                                     (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      ATGCTTCCGGCTCGTATG18                                                          (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 90 AMINO ACIDS                                                    (B) TYPE: AMINO ACID                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: PROTEIN                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      MetGlySerAlaGlyAlaArgProAlaLeuAlaAlaAlaLeuLeu                                 51015                                                                         CysLeuAlaArgLeuAlaLeuGlySerProCysProAlaValCys                                 202530                                                                        GlnCysProAlaAlaAlaProGlnCysAlaProGlyValGlyLeu                                 354045                                                                        ValProAspGlyCysGlyCysCysLysValCysAlaLysGlnLeu                                 505560                                                                        AsnGluAspCysSerArgThrGlnProCysAspHisThrLysGly                                 657075                                                                        LeuGluCysAsnArgLeuValAsnAspIleHisLysPheArgAsp                                 808590                                                                        (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 90 AMINO ACIDS                                                    (B) TYPE: AMINO ACID                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: PROTEIN                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      MetSerSerSerThrPheArgThrLeuAlaValAlaValThrLeu                                 51015                                                                         AlaHisLeuThrArgLeuAlaLeuSerThrCysProAlaAlaCys                                 202530                                                                        HisCysProLeuGluAlaProLysCysAlaProGlyValGlyLeu                                 354045                                                                        ValArgAspGlyCysGlyCysCysLysValCysAlaLysGlnLeu                                 505560                                                                        AsnGluAspCysSerLysThrGlnProCysAspHisThrLysGly                                 657075                                                                        LeuGluCysAsnSerLeuPheAsnAspIleHisLysPheArgAsp                                 808590                                                                        (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 93 AMINO ACIDS                                                    (B) TYPE: AMINO ACID                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: PROTEIN                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      MetThrAlaAlaSerMetGlyProValArgValAlaPheValVal                                 51015                                                                         LeuLeuAlaLeuCysSerArgProAlaValGlyGlnAsnCysSer                                 202530                                                                        GlyProCysArgCysProAspGluProAlaProArgCysProAla                                 354045                                                                        GlyValSerLeuValLeuAspGlyCysGlyCysCysArgValCys                                 505560                                                                        AlaLysGlnLeuGlyGluLysCysThrGluArgAspProCysAsp                                 657075                                                                        ProHisLysGlyLeuPheCysAspTyrTyrArgLysMetTyrGly                                 808590                                                                        AspMetAla                                                                     (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 51 AMINO ACIDS                                                    (B) TYPE: AMINO ACID                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: PROTEIN                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      MetLeuAlaSerValAlaGlyProIleSerLeuAlaLeuValLeu                                 51015                                                                         LeuAlaLeuCysThrArgThrAlaThrGlyGlnAspCysSerAla                                 202530                                                                        GlnCysGlnCysAlaAlaGluAlaAlaProHisTyrTyrArgLys                                 354045                                                                        MetTyrGlyAspMetAla                                                            50                                                                            (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 54 AMINO ACIDS                                                    (B) TYPE: AMINO ACID                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: PROTEIN                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      MetGluThrGlyGlyGlyGlnGlnLeuProValLeuLeuLeuLeu                                 51015                                                                         LeuLeuLeuLeuArgProCysGluValSerGlyArgGluAlaAla                                 202530                                                                        CysProArgProCysGlyGlyArgCysProAlaGluProProArg                                 354045                                                                        AspProMetSerSerGluAlaLysIle                                                   50                                                                            (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 50 AMINO ACIDS                                                    (B) TYPE: AMINO ACID                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: PROTEIN                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      MetGlnArgAlaArgProThrLeuTrpAlaAlaAlaLeuThrLeu                                 51015                                                                         LeuValLeuLeuArgGlyProProValAlaArgAlaGlyAlaSer                                 202530                                                                        SerGlyGlyLeuGlyProValValArgCysGluProCysValAla                                 354045                                                                        ArgAlaLeuAlaArg                                                               50                                                                            (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 AMINO ACIDS                                                    (B) TYPE: AMINO ACID                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: PROTEIN                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      MetLysSerValLeuLeuLeuThrThrLeuLeuValProAlaHis                                 51015                                                                         LeuValAlaAlaTrpSerAsnMetTyrAlaValAspCysProGln                                 202530                                                                        HisCysAspSerSerGluCysLysSerSerProArg                                          3540                                                                          __________________________________________________________________________

We claim:
 1. An isolated polynucleotide comprising a polynucleotide having at least 95% identity to a member selected from the group consisting of:(a) a polynucleotide encoding a polypeptide comprising the amino acid sequence from amino acid 1 to 163 of SEQ ID NO:2; and (b) the complement of (a).
 2. The isolated polynucleotide of claim 1 wherein said member is (a).
 3. The isolated polynucleotide of claim 2, wherein the polynucleotide is DNA.
 4. A method of making a recombinant vector comprising inserting the isolated polynucleotide of claim 2 into a vector, wherein said polynucleotide is DNA.
 5. A recombinant vector comprising the polynucleotide of claim 2, wherein said polynucleotide is DNA.
 6. A recombinant host cell comprising the polynucleotide of claim 2, wherein said polynucleotide is DNA.
 7. A method for producing a polypeptide comprising expressing from the recombinant cell of claim 6 the polypeptide encoded by said polynucleotide; said polypeptide having the ability to stimulate proliferation of endothelial cells in the presence of comitogen Con A.
 8. The isolated polynucleotide of claim 1 wherein said member is (a) and the polypeptide comprises amino acids -21 to 163 of SEQ ID No:2.
 9. The isolated polynucleotide of claim 1 comprising a polynucleotide encoding a polypeptide comprising the amino acid sequence identical to amino acids 1 to 163 of SEQ ID NO:2.
 10. A process for producing a polypeptide comprising:expressing from a recombinant cell containing the polynucleotide of claim 9 the polypeptide encoded by said polynucleotide.
 11. The isolated polynucleotide of claim 9 comprising nucleotides 121 to 609 of SEQ ID NO:1.
 12. The isolated polynucleotide of claim 9 comprising nucleotides 58 to 609 of SEQ ID NO:1.
 13. The isolated polynucleotide of claim 9 comprising nucleotides 1 to 1271 of SEQ ID NO:1.
 14. The isolated polynucleotide of claim 1 comprising a polynucleotide encoding a polypeptide comprising the amino sequence identical to amino acids -21 to 163 of SEQ ID NO:2.
 15. A method for producing a polypeptide comprising:expressing from a recombinant cell containing the polynucleotide of claim 14 the polypeptide encoded by said polynucleotide; said polypeptide having the ability to stimulate proliferation of endothelial cells in the presence of comitogen Con A.
 16. The isolated polynucleotide of claim 1, wherein said polynucleotide is RNA.
 17. An isolated polynucleotide comprising a polynucleotide having at least a 95% identity to a member selected from the group consisting of:(a) a polynucleotide encoding the same mature polypeptide encoded by the human cDNA in ATCC Deposit No. 75874; and (b) the complement of (a).
 18. The isolated polynucleotide of claim 17, wherein the member is (a).
 19. A method for producing a polypeptide comprising:expressing from a recombinant cell containing the polynucleotide of claim 18 the polypeptide encoded by said polynucleotide; said polypeptide having the ability to stimulate proliferation of endothelial cells in the presence of comitogen Con A.
 20. The isolated polynucleotide of claim 19, wherein said polynucleotide comprises DNA identical to the coding portion of the human cDNA in ATCC Deposit No. 75874 which encodes a mature polypeptide.
 21. A process for producing a polypeptide comprising:expressing from a recombinant cell containing the polynucleotide of claim 20 the mature polypeptide encoded by said polynucleotide.
 22. A method for producing a polypeptide comprising:expressing from a recombinant cell containing the polynucleotide of claim 17 the polypeptide encoded by said polynucleotide; said polypeptide having the ability to stimulate proliferation of endothelial cells in the presence of comitogen Con A. 